172-Estradiol (E2) has been implicated to exert neuroprotection in a variety of neurodegenerative disorders, including stroke; however, the mechanisms underlying its nongenomic and genomic signaling in the brain, and its neuroprotective effects remains unclear. Work by our group may shed light on this issue via our cloning of a novel ER coregulator, called PELP1, which we propose is the critical missing link that explains E2 ability to induce both nongenomic and genomic signaling in the brain and neuroprotection. To test our hypothesis, Aim 1 would use a PELP1 forebrain-specific KO (PELP1 FB KO) mouse model to determine the role of PELP1 in E2 nongenomic and genomic signaling, antioxidant actions, and neuroprotective effects in the brain following cerebral ischemia. Since little is known about the regulation of PELP1 in the brain, Aim 2 would characterize PELP1 expression, phosphorylation and signalsome formation in the brain following cerebral ischemia, determine the regulatory role of E2, and identify kinases responsible for the phosphorylation of PELP1. Preliminary data suggest that PELP1 may also play an important role in regulating local E2 production in the brain by regulating activation of the brain aromatase promoter. Thus, Aim 3 would examine the effect of PELP1 knockout on basal and E2-induced aromatase expression and activity in the brain through use of PELP1 FB KO mice, and identify the specific brain aromatase promoter regulated by PELP1. Recruitment of PELP1 to the brain aromatase promoter would also be assessed by ChIP, and the potential role of local E2 production in amplifying neuroprotection by low physiological levels of E2 would also be examined. Finally, Aim 4 would test the hypothesis that loss of E2 neuroprotective ability after a period of long-term E2 deprivation (such as occurs after menopause) is due to a brain-specific epigenetic gene silencing of PELP1 and/or ER1, and would determine whether the gene silencing is reversible and whether E2 sensitivity can be reinstated in the brain. The proposed studies have the potential to significantly advance our understanding of how E2 exerts its signaling and neuroprotective effects in the brain, and may provide a mechanistic understanding of why E2 failed to exert beneficial cardiovascular and neural effects in the WHI study, where E2 replacement was begun long after the onset of menopause.